Research Paper Volume 3, Issue 5 pp 515—532

Figure 6. M1775R is defective in CtIP binding and increases RAD51 and RPA pan-nuclear immuno-staining that in part co-localizes with elevated ssDNA formation but not atypical PML-NBs. (A) HEK293T cells were co-transfected with Myc-CtIP plasmid and the indicated FLAG-BRCA1 plasmids. Immuno-precipitated FLAG material was examined for CtIP binding by western blot analysis. (B) RAD51 staining of unirradiated HCC1937 cells infected with the indicated HD-Ad vectors. (C) RPA staining of representative HCC1937 cells treated as in B. The percentage of cells having large, clustered RPA foci was <1% for wild-type BRCA1 and 7-10% for M1775R. (D) Western blot analysis for FLAG-BRCA1 expression in infected HCC1937 cells with DNA-PKcs as loading control. (E) RPA and ssDNA co-staining of representative HCC1937 cell nuclei treated as in B and C. Cells labeled in parallel with BrdU were denatured to determine the extent of incorporation. (F) RPA and PML co-staining of representative HCC1937 cell nuclei treated as in B, C, and E. The percentage of cells having thread-like PML-NBs was <1% for wild-type BRCA1 and >50% for M1775R.